Project Summary/Abstract Obesity-induced lipotoxicity is the primary pathophysiological defect that predisposes to type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). Because current management options remain limited, identification of new regulatory mechanisms that govern the maladaptive metabolic response to excess lipids should serve to identify novel opportunities for pharmacologic intervention. The long-term objective of this research is to define a novel molecular mechanism by which excess fatty acids impair lipid and glucose homeostasis. The rationale is that the identification of a novel mechanism that promotes lipotoxicity could lead to new therapeutic targets. Our preliminary studies indicate that acyl-CoA thioesterase 9 (Acot9), an enzyme that deactivates fatty acids in vitro, promotes adiposity and hepatic glucose production (HGP) in mice in the setting of excess lipid uptake. Guided by extensive preliminary data, the central hypothesis of this research plan is that, in response to overnutrition, Acot9-mediated regulation of lipid metabolism impairs hepatic insulin signaling and exacerbates HGP. This will be tested in two specific aims: 1) Identify the molecular mechanisms by which Acot9 regulates hepatic lipid and glucose metabolism; 2) Determine the molecular mechanisms by which high fat diet regulates the subcellular localization and the thioesterase activity of Acot9. In aim 1, the effect of Acot9 expression on glucose homeostasis will be determined in mice with systemic and liver-specific ablation of Acot9 by hyperinsulinemic/euglycemic clamp. Liver histology, mass spectrometry of lipidomics and established assays to measure lipid levels will be used to characterize the effect of Acot9 on lipid metabolism. VLDL secretion will be assessed following Tyloxapol injection. ?-oxidation will be determined using radiolabeled palmitate and Seahorse flux analyzer. Fatty acid uptake, efflux, lipogenesis and lipolysis will be studied in primary adipocytes using radiolabeled palmitate or acetate. Immunoblot and qPCR analyses will measure established regulators of insulin signaling, HGP and lipid homeostasis. It is anticipated that Acot9 will promote insulin resistance and HGP by exacerbating the formation of lipotoxic intermediates such as reactive oxygen species, ceramides and diacylglycerol and by increasing ?-oxidation. Aim2 will test whether high fat diet- induced expression and phosphorylation of Acot9 alters the subcellular localization, substrate specificity and activity of Acot9. This will be tested in palmitate-treated cells using microscopy and in thioesterase activity assays using recombinant Acot9. We expect that excessive nutrition will increase localization of Acot9 to ER and its affinity for saturated fatty acyl-CoA. Overall, this proposal will elucidate mechanisms by which Acot9 promotes lipotoxicity, which is significant because trafficking of fatty acids varies in health and disease. These studies are expected to identify new therapeutic targets for the management of type 2 diabetes and NAFLD. Baran A. Ersoy, Ph.D.